DESCRIPTION: Deficiency of fumarylacetoacetate hydrolase (FAH) causes tyrosinemia type I (hepatorenal tyrosinemia, HT1) in humans and is characterized by severe liver dysfunction, renal tubular damage and liver cancer. In the murine model of HT1 hepatocytes stably expressing Fah have a strong selective growth advantage. Fah mutant mice are therefore uniquely suited for the study of the biology of integrating gene therapy vectors in the liver. Recently, we have found that in vivo selection of Fah+ cells also occurs in the renal proximal tubular epithelium. Therefore, HT1 mice are well suited to study hepatic and renal gene therapy with integrating vectors. In the past funding period, we were able to demonstrate gene repair in hepatocytes and renal proximal using AAV2/2 and AAV2/8 vectors harboring 4 kb of genomic homology to an Fah point mutation mouse. In addition, we developed a novel AAV vector designed to integrate cDNA expression cassettes into the ribosomal DNA rDNA) locus by homologous recombination. We found site-specific integration in vivo in multiple tissues including liver, kidney, heart, lung and skeletal muscle. We also succeeded in the synthesis of a potent small molecule inhibitor of Fah (CEHPOBA), which works in vivo. Because of the powerful selection found in genetic Fah deficiency, pharmacological inhibition of Fah may be a general strategy for the in vivo selection of genetically modified hepatocytes. We have demonstrated that hepatocytes deficient for homogentisic acid dioxygenase (Hgd) could be positively selected in vivo after transplantation into CEHPOBA treated mice. The overall theme of this application is the utilization of the tyrosine catabolic pathway for in vivo metabolic selection of genetically modified epithelial cells in the liver and kidney. Specifically , we will use the model to further develop AAV-based vectors for in vivo gene repair and targeted site-specific integration. We will also explore the use of CEHPOBA for in vivo selection of transgene modified hepatocytes. Public Health Relevance: This research investigates novel strategies of general significance for gene therapy of many genetic hepatic and renal disorders. Improved AAV vectors for gene repair as well as site-specific chromosomal integration will be generated. In addition, a broadly applicable platform for pharmacological growth selection of genetically modified hepatocytes is being developed.